The Mechanism And Intervention Of Glucocorticoids Inhibiting Airway Epithelial Cells Repair In Balb/C Mouse

Part Ⅰ Epithelial Repair and AHR in an Asthma Mice Model and the Effect of Short-term Dexamethasone TreatmentOBJECTIVE:To observe glucocorticoid dexamethasone (DEX)-induced modifications in asthmatic airway epithelium and investigate the resultant consequences; to analyze the relationships between airway hyperresponsiveness (AHR), airway neutrophils, and shed airway epithelial cells in this pathogenic process.METHODS:BALB/c mice were divided into four groups:naive, asthma, asthma+DEX1and asthma+DEX5. The latter three groups were used to recapitulate an asthma model. The treatments asthma+DEX1and asthma+DEX5received DEX by intraperitoneal injection. Within24hours after the final challenge, the mice were examined to determine development of AHR; bronchoalveolar lavage fluid (BALF) was obtained and total cells present in the BALF were counted and classified. Lung and trachea pathology was determined by paraffin embedded sectioning with hematoxylin-eosin staining, anti-Ki-67and anti-caspase3immunohistochemical staining.RESULTS:Short-term DEX treatment exacerbated the asthmatic disruption of tracheal epithelium and appeared to have a limited effect on relieving AHR, although it led to decreased counts of neutrophils and shed airway epithelial cells. Immunohistochemical staining showed that DEX treatment prevented asthmatic airway epithelial cell division, as evidenced by anti-Ki-67staining, but had a limited effect on apoptosis signaling, as evidenced by caspase-3staining. The counts of neutrophils and shed epithelial cells were strongly correlated with each other and each with Penh50values.CONCLUSION:Short-term DEX treatment decreases asthmatic airway epithelial loss to a minor extent but significantly suppresses airway epithelial proliferation. These results provided evidence of defects in airway epithelial expansion. Additionally, airway neutrophil accumulation might exacerbate airway epithelial injury. These processes may represent important factors underlying development of AHR in asthma. Part Ⅱ Effect of Vitamin A on Proliferation and Apoptosis of Asthmatic Airway Epithelial Cells and Airway InflammationOBJECTIVE:To observe the effect of Vit A on asthmatic airway epithelial integrity, and the influence of Vit A on asthmatic inflammation.METHODS:BALB/c mice were divided into four groups:naive, asthma, asthma+VitA25and asthma+VitA250. The latter three groups were used to recapitulate an asthma model. Within24hours after the final challenge, the mice were examined to determine development of AHR; bronchoalveolar lavage fluid (BALF) was obtained and total cells present in the BALF were counted and classified. Lung and trachea pathology was determined by paraffin embedded sectioning with hematoxylin-eosin staining, anti-Ki-67and anti-caspase3immunohistochemical staining. Cytokine in BALF also were detected by ELISA.RESULTS:Vitamin A decreased shed epithelial cells and neutrophils in asthmatic airway, and also down-regulated airway TGF-β1. Vitamin A significantly prohibited airway epithelial apoptosis detected by anti-caspase3staining and down-regulated AHR significantly. Anti-Ki-67stain did not indicate the acceleration of Vit A on asthmatic airway epithelial cells. The counts of neutrophils were correlated with shed airway epithelial cells and Penh50values.CONCLUSION:Vitamin A significantly decreased asthmatic epithelial injury and down-regulated AHR and airway neutrophil accumulation. However, whether Vit A promoted proliferation was still uncertain. Airway neutrophil accumulation might led asthmatic airway epithelial injury and be a closely related factor to lead AHR. Part III Effect of DEX Combined with GILZ Silence and Vitamin A on Airway Epithelium and Inflammation in a BALB/c Asthmatic Mice ModelOBJECTIVE:To observe whether GILZ silence or Vit A could reverse adverse effect of DEX on asthmatic epithelial cells.METHODS:GILZ silence adenoviruses or no-load adenoviruses were to infect BALB/c mice4days before OVA challenge. Within24hours after the final challenge, the mice were examined to determine development of AHR; bronchoalveolar lavage fluid (BALF) was obtained and total cells present in the BALF were counted and classified. Lung and trachea pathology was determined by paraffin embedded sectioning with hematoxylin-eosin staining, anti-Ki-67and anti-caspase3immunohistochemical staining.RESULTS:DEX combined with GILZ silence or Vit A decreased shed asthmatic airway epithelial cells, and DEX combined with Vit A decreased asthmatic AHR. DEX combined with Vit A inhibited airway epithelial apoptosis significantly and combined with GILZ silence down-regulated slightly airway epithelial apoptosis. No clear evidence showed any promotional effect on asthmatic epithelial proliferation after DEX combined with GILZ silence or Vit A treatment.CONCLUSION:Vitamin A and GILZ silence both decrease asthmatic airway epithelial injury, and the effects of former two on airway epithelial proliferation are still unclear. Part IV Effects of DEX, Vit A and GILZ Silence on EGFR/Raf-1/MEK/Erk Signal Pathway in Asthmatic Mouse AirwayOBJECTIVE:To observe the influnce of DEX and Vit A on the EGFR/Raf-1/MEK/Erk signal pathway. To observe DEX and Vit A regulate EGFR/Raf-1/MEK/Erk pathway by GILZ.METHODS:mRNA was extracted from mouse lungs and then was used to synthesize cDNA. qPCR analysis was used to detect transcriptional activity in EGFR/Raf-1/MEK/Erk signal pathway. Protein was extracted from mouse lungs, Western Blot analysis was used to detect the expression and activation of factors in EGFR/Raf-1/MEK/Erk signal pathway.RESULTS:DEX and Vit A alerted the transcriptional activity of Raf-1, MEK1/2and Erkl/2detected by qPCR. IHC-P showed DEX induced GILZ expression and more dosage of DEX induced more GILZ; Vit A had no significant effect on GILZ expression; DEX down-regulated Erkl/2expression and combined with Vit A maintained higher expression of Erk1/2; Erkl/2expression was enhanced after GILZ silence.CONCLUSION:DEX was enrolled in the regulation of Raf-1/MEK/Erk signal pathway by induced GILZ, and more dosage of DEX more negative regulation. Vit A maintain the activity of Raf-1/MEK/Erk signal pathway to some extent, and its mechanism might be the enhance activity of factors in the signal way by Vit A, although lack of full experiment.